The goal of this application is to develop a high throughput screening assay to identify fibrin-specific compounds. The ultimate goal is to develop new molecular probes to image fibrin, its formation, and its dissolution. Fibrin is a major component of thrombus (blood clot), and thrombus is implicated in a range of pathologies, e.g. ischemic stroke, myocardial infarction, pulmonary embolism, and deep vein thrombosis. Beyond hemostasis, it is known that most solid tumors are characterized by the presence of fibrin clots. More recently fibrinogen and fibrin have been implicated in multiple sclerosis. Imaging of fibrin using a fibrin-specific molecule conjugated to an optical, magnetic resonance (MR), or radioactive reporter could provide insights into the biology of all these diseases. Fibrin probes could be used to identify thrombi or tumor metastases and guide patient management. We have worked with two families of peptides that bind to two separate sites on fibrin, but do not bind to fibrinogen, the soluble precursor protein to fibrin. We have synthesized these peptides conjugated to optical, MR, and/or radioactive tags for imaging. However the peptides are unstable in the body and are rapidly degraded by peptidases in the liver and kidneys. This rapid in vivo metabolism limits the utility of such probes. We have shown that these peptides also bind to a soluble fibrin degradation product, DD(E), and that a competitive binding assay can be implemented. The assay involves displacement of a fluorescently labeled peptide from DD(E) by a competitor ligand. Using polarized fluorescence, the anisotropy of the protein-bound fluorescent probe is high, but is much lower when it is displaced. The assay is solution phase and can be run in a microtiter plate format in a simple mix and measure format. We have also shown that both classes of fibrin binding peptide can be analyzed simultaneously, simply by using a different fluorophore on each peptide. In this application we propose to further develop this assay for translation to an automated HTS environment. We will also develop secondary screens to confirm compounds active in the primary screen as reproducible hits, and rule out artifacts, as well as develop counter-screening assays to allow prioritization of hits for further testing. The result of this work will be a validated HTS assay for fibrin binders that will be submitted to the Molecular Libraries Probe Production Centers Network for screening with their libraries to identify new potent and selective fibrin binders.